An additional methyl group at the 10-position of retinal dramatically slows down the kinetics of the rhodopsin photocascade.
The present study focuses on ligand-protein interactions in a rhodopsin analogue generated from bovine opsin and the 10-methyl homologue of 11-cis-retinal. The analogue pigment displays a reduced alpha-band at 506 +/- 2 and a stronger beta-band at 325 nm. Remarkably, the rotational strength of these bands observed in visible circular dichroism spectra was found to be similar for both native and 10-methyl rhodopsin. The quantum yield of the analogue pigment was determined to be 0.55. All photointermediates were analyzed by Fourier transform infrared difference spectroscopy. At the batho stage, strong hydrogen-out-of-plane vibrations were observed, indicating that the 10-methyl chromophore also adopts a distorted all-trans conformation at this stage. In contrast to native rhodopsin, the batho intermediate of the 10-methyl pigment is stable up to 180 K and only slowly decays to the next intermediate between 180 and 210 K. As in native rhodopsin, the 10-methyl metarhodopsin I intermediate is generated at about 220 K, but its transition to the metarhodopsin II state is again shifted to a much higher temperature (> 293 K) than for the native pigment (> 260 K). Infrared analysis, nevertheless, shows that the conformational changes in the photointermediates of the 10-methyl pigment are basically identical with those observed in the native pigment. This is supported by a signal function assay, showing that the analogue pigment is able to activate transducin. The dual effect of the 10-methyl group on the photocascade is attributed to steric interactions which, initially, hamper the relaxation of strain in the polyene chain of the chromophore and, eventually, interfere with the conformational rearrangements of the protein moiety required to adopt the active conformation of the receptor. Our data provide direct support for the concept that the relaxation of strain in the retinal polyene chain acts as the major driving force of the photocascade dark reaction.
DeLange
F
F
Department of Biochemistry FMW-160, University of Nijmegen, The Netherlands.
Bovee-Geurts
P H
PH
VanOostrum
J
J
Portier
M D
MD
Verdegem
P J
PJ
Lugtenburg
J
J
DeGrip
W J
WJ
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D007700:Kinetics; D010782:Photolysis; D012172:Retinaldehyde / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D017299:Rod Opsins / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D015293:Transducin / Q000378:metabolism
D017299:Rod Opsins; D012243:Rhodopsin; D015293:Transducin; D012172:Retinaldehyde